Nerve cells (neurons) function as input and output devices. Each neuron makes specific connections with other cells both to receive signals and to transmit impulses to postsynaptic partners. The mechanisms that control the fidelity of these choices are largely unknown but are likely to depend on specific gene regulation. We have shown that the UNC-4 homeodomain protein and its Groucho-like transcriptional co-repressor protein, UNC-37, are required in the VA class of C. elegans motor neurons to specify synapses to these cells. In unc-4 and unc-37 mutants, the Vas adopt inputs normally reserved for VB motor neurons. Genes that UNC-4 and UNC-37 repress may encode molecules that induce the creation of these ectopic connections. A major goal of this project is to identify these unc-4 target genes. Our strategy will exploit C. elegans primary cell culture methods, newly developed in this laboratory, to isolate GFP-labeled motor neurons in which unc-4 functions. Microarray experiments with cDNA probes prepared from these cells should reveal unc-4 regulated genes. We will take advantage of the genetic tractability of C. elegans to inactivate these genes and thereby test them for roles in synaptic choice. Potential wiring defects in these mutants will be assayed by serial section electron microscopy and with optical methods that rely on labeling presynaptic and postsynaptic proteins with different-colored GFPs. The DNA sequences of these unc-4 target genes could be used to identify similar genes in mammals where their roles in neuronal synaptogenesis can be explored. Thus, our work with a simple nematode model system is likely to provide important clues to fundamental processes governing the development and function of the human brain.